One of the more serious diseases of infancy is whooping cough, which is an acute, highly communicable, infectious disease characterized by a paroxysmal or spasmodic cough which usually ends with a prolonged, high-pitched crowing inspiration (the whoop). In infants, choking spells may be more common than whoops. The disease is caused by the organism Bordetella pertussis. Immunization against this disorder has been accomplished in the past by injection of either killed-cell vaccine or extracted antigen. Until the present time, however, the available preparations, which contain a large amount of bacterial cells, have had certain shortcomings. In particular, such preparations have a tendency to produce side reactions such as fever, irritation, redness and the like.
Currently, B. pertussis vaccine is composed of the whole bacteria of B. pertussis, or of the whole bacteria and an adjuvant such as aluminum phosphate, aluminum hydroxide or a mixture thereof. A description of said vaccine production may be found in U.S. Pat. Nos. 3,577,319 and 4,429,046 and in the concurrently filed patent application of Wenlii Lin and William A. Griffith entitled: "Enhanced Large Scale Cultivation of Bordetella Pertussis Cells for Vaccine Production", the teachings of which are incorporated herein by reference.
Japanese Pat. No. 55-141416, entitled: "A Triple Vaccine Prepared by Adding Diphtheria Toxoid and Tetanus Toxoid to a Pertussis HA Fraction," employs the use of sucrose gradient centrifugation to remove endotoxin from Phase I B. pertussis hemagglutinin (HA) fraction after precipitating the culture filtrate with ammonium sulfate, then detoxifying the histamine-sensitizing factor (HSF) and leukocytosis promoting factor (LPF) in the HA fraction thus obtained. The shortcoming of this technology is its low capacity and high time consumption. Thus, it would require twenty hours to process up to 200 ml of a preparation using the largest Beckman zonal centrifuge.
U.S. Pat. No. 3,897,309 describes the removal of trace amounts of pyrogens from aqueous solutions by passing a strongly ionic solution thereof through a column of a basic anion exchange resin. The invention describes a specific process for the removal of pyrogens from L-asparaginase. When impure L-asparaginase is dissolved in a strongly ionic buffer solution and passed through a column of a basic anion exchange resin the pyrogens are selectively and irreversibly adsorbed onto the anion exchange resin while the L-asparaginase solution, free of pyrogens passes through the column. Examples of the resins cited in said patent are: Sephadex.RTM. DEAE-A-25, Sephadex.RTM. DEAE-A-50, Dowex.RTM. 1X2, Amberlite.RTM. IRA-938 and Whatman DE-52.
The chemical properties of endtoxins and proteins of different sources vary greatly, thus making it impossible for those familiar with the art to anticipate from the immediately preceding patent that a particular ion-exchange resin can separate endotoxin from the active components of B. pertussis vaccines. In fact, when the procedure of Example 4 in the above last cited patent was studied, using DE-52 resin, it was concluded to be inapplicable to B. pertussis vaccine preparation.
It is an object of the present invention to provide a B. pertussis vaccine of substantially improved form, having a markedly reduced tendency to produce side effects, while providing long-term immunity against whooping cough.